https://doi.org/10.5812/zjrms.88964

Detection of Entamoeba histolytica Coproantigen Among Children with Dysentery in Ahvaz, Southwest Iran

authors:

avatar Ahmad Shamsizadeh 1 , avatar Roya Nikfar 2 , avatar Mahmoud Rahdar ORCID 1 , *

Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Abuzar Children’s Hospital, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Zahedan Journal of Research in Medical Sciences: Vol.22, issue 3; e88964
published online: August 4, 2020
article type: Research Article
received: January 12, 2019
revised: September 4, 2019
accepted: September 28, 2019

how to cite: Shamsizadeh A , Nikfar R, Rahdar M. Detection of Entamoeba histolytica Coproantigen Among Children with Dysentery in Ahvaz, Southwest Iran. Zahedan J Res Med Sci. 2020;22(3):e88964. https://doi.org/10.5812/zjrms.88964.

Abstract

Entamoeba histolytica is one of the important parasitic diseases in many parts of the world, especially the tropical and subtropical regions. The parasite is transmitted through contaminated water and vegetables. The exact diagnosis of infection with the parasite is crucial in many medical laboratories since there are many false positive and negative results in their reports. Therefore, the current study aimed at evaluating and comparing microscopic and coproantigen ELISA (the enzyme-linked immunosorbent assay) results to reach an appropriate test for the correct diagnosis of amoebiasis in children. One hundred stool samples were collected from children under 15 years old with dysenteric diarrhea from April to September 2018. Microscopic tests and coproantigen ELISA were performed on all the samples. The results showed that 5% of the samples had E. histolytica/E. dispar cysts. The findings of ELISA to detect coproantigen did not show any specific E. histolytica antigen in the samples. Hence, all the patients received chemotherapy for shigellosis. E. histolytica infection is not the main causative agent for dysenteric diarrhea in children in the studied area, and laboratory experts should be trained to prevent false-positive reports.

1. Background

Entamoeba histolytica is a pathogen parasite causing acute enteritis with dysenteric symptoms in susceptible individuals. The parasite spreads to other parts of the body via the bloodstream and causes amoebic abscesses in the liver, lung, brain, and skin (1). Approximately 10% of the world population (more than 500 million) are infected with E. histolytica, 1% of which develop the invasive form of the disease with an annual death toll of 100,000 (2). Amoebiasis is the leading cause of death from a parasitic infection after malaria in the world with the annual death toll of 100,000 in the tropical areas and developing countries (3, 4). According to microscopic studies, the infection's prevalence is 2.2% to 30% in different parts of Iran (5). Some other studies showed that the prevalence of E. histolytica and E. dispar in Central, Northern, and Southern parts of Iran were 0.78%, 3.9%, and 4.6%, respectively (6). There are three other non-pathogenic species of Entamoeba- i.e., E. dispar, E. moshkovskii, and the recently described E. bangladeshi, which their trophozoites and cysts are morphologically very similar to those of E. histolytica (7). One of the important views on the diagnosis of the parasite is the establishment of precise, rapid, and accessible diagnostic tests in all laboratory centers. There are several methods to detect E. histolytica in serum and stool, including microscopic detection of cysts and trophozoites in stool samples, as well as coproantigen detection, PCR, and antibody detection in sera (7). Microscopic examination has a low sensitivity (60%) due to the difficulty of distinguishing between pathogenic and non-pathogenic Entamoeba species and the lack of expert practitioners in many laboratories (8). Also, the E. histolytica cyst is not easily differentiated from white blood cells in stool samples (9). Antibody detection in serum samples of the infected patients is not reliable since only 70% - 80% of patients are seropositive, and, on the other hand, more than 25% of people are seropositive in endemic areas (10, 11). Real-time PCR and isoenzyme analysis of culture are two sensitive methods to distinguish the pathogen from nonpathogen species, but they are not practical in many laboratories due to lack of expert staff, high cost, and their time-consuming nature (8, 12). According to the World Health Organization recommendations, an accessible, rapid, and sensitive test should be considered for the more precise diagnosis of E. histolytics, especially in developing countries (1, 13). The proper diagnosis of E. histolytica in patients is of great importance to prevent unnecessary treatment and apply appropriate drugs (14). The detection of E. histolytica antigen in the stool (coproantigen) by the ELISA (enzyme-linked immunosorbent assay) technique is easy, rapid, and more sensitive than microscopic tests and is practical in all diagnostic laboratories (5, 10, 15). Distinguishing E. histolytica from other causes of dysentery, such as shigellosis, is a critical problem in many diagnostic laboratories.

2. Objectives

Therefore, the current study aimed at differentiating E. histolytica from shigellosis by detecting coproantigen in stool samples of children with dysentery for the proper diagnosis and treatment.

3. Methods

Totally, 100 stool samples were collected from children under 15 with dysenteric diarrhea in a pediatric hospital from April to September 2018. This project was approved by the Ethics Committee of Ahvaz Jundishpur University of Medical Sciences (code no.: ajums REC.1393014). The samples were transferred to the Parasitology Department of Medical School and microscopically examined for the detection of trophozoites or cysts of E. histolytica. The samples were stained with trichrome staining methods and examined for trophozoites and cysts of E. histolytica. Horse albumin was used to fix stool smears. After microscopic examination, the samples were stored at -20°C until used for ELISA (Biomerica, Germany catalog no. 7078). The kit had been coated with a monoclonal antibody against E. histolytica specific antigen (ESA), and after adding the sample, the other antibody against E. histolytica was used (sandwich ELISA). Tetramethyl benzidine and peroxidase were used as the substrate and enzyme to create a yellow-color complex. The optical density was read at the wavelength of 450 nm by the ELISA reader machine. According to the instructions of the kit manufacturer, at the wavelength of 450 nm, the positive control should be greater than 0.5 and the negative control less than 0.15 OD. The borderline for positive or negative samples was 0.15 OD.

4. Results

The current study results indicated that the prevalence of this disease in Iran, as mentioned in the introduction, should be amended and updated. Microscopic examination of samples revealed that 5% (five out of 100) of stool samples contained E. histolytica/E. dispar cysts. The results of the ELISA showed that none of the stool samples were positive, and the OD of all samples was under 0.15.

5. Discussion

The current study results showed no positive samples for E. histolytica infection among 100 child patients with dysenteric diarrhea, using the coproantigen ELISA test. The prevalence of infection was previously investigated. In a two-year study, Safi et al. (16), reported that 1.83% out of 14,614 patients with gastrointestinal (GI) symptoms were infected with E. histolytica/E. dispar. Yosefi et al. (17), showed that 1.7% out of 100 samples obtained from HIV+ patients contained E. histolytica cysts. Rafiei et al. (18), studied the contamination of surface waters and presented that 50% of 44 river and surface water samples from Ahvaz and 6.3% of water samples from Shush, Khuzestan Province, Southwest Iran, were contaminated with Entamoeba spp. (19).

The prevalence of intestinal parasite was also studied in other parts of Iran. Zebardast et al. (9), reported that 153 stool specimens out of 1520 (10%) were infected with intestinal parasites in patients with GI disorders in Tehran. The parasites included Blastocystis spp. (4.73%), Giardia intestinalis (2.30%), E. coli (1.38%), Endolimax nana (0.92%), Cryptosporidium spp. (0.06%), E. dispar (0.06%), Dientamoeba fragilis (0.06%), Iodamoeba butschlii (0.06%), Chilomastix mesnili (0.06%), Hymenolepis nana (0.19%), and Dicrocoelium dendriticum (0.13%). They did not observe E. histolytica in any of the specimens. The prevalence of E. dispar is 10 times more than E. histolytica, and it is estimated that E. dispar is the main enteric amoeba in the Central and Northern areas of Iran. Feiz Haddad et al. (20), showed that 10.68% of stool samples referred to the central laboratory of Dezful contained E. histolytica/E. disper. It seems that the E. histolytica infection has a rare prevalence in many parts of Iran (21). Solaymani et al. (22), along with other researchers, confirmed that all Iranian asymptomatic cyst passers were infected with E. dispar; a nonpathogen species of Entamoeba genus (6, 22, 23). Savadkoohi et al. (24), reported that 6% out of 537 children with dysenteric symptoms presented E. histolytica infection with positive microscopic stool examinations in Babol City, North of Iran. The prevalence of E. histolytica infection was 1% among patients with GI diseases in Tehran hospitals (25). A meta-analysis on findings from 1988 to 2009 estimated the average prevalence of 1.3% for E. histolytica in Iran. This finding indicated a recently decreased infection rate of E. histolytica in many parts of Iran. Promotion of a healthy lifestyle, the increase of people’s health knowledge, and the consumption of safe water in many parts of the country are the main reasons for decreasing E. histolytica infection in Iran (26). Reports from different parts of the world indicate the infection rate of E. histolytica as 10.6% in Jordan (27), 11% in New Delhi (28), 5.3% in Turkey (29), 9.2% in Saudi Arabia (30), 65.7% in Nigeria (31), and 66.6 in Nepal (32).

In many medical laboratories, E. histolytica in stool specimens is commonly diagnosed by a direct microscopic examination in order to save time and expenditure, and ease of performance. Unlike helminths, the direct method is not suitable for the diagnosis of protozoan infection; the idea confirmed by many studies. The technical level and the experience of the laboratory practitioner, as well as useful training, are the crucial criteria for proper identification of protozoa in stool samples (33-35). Uslu et al. (14), reported that the direct microscopic method provides false-positive results in half of the patients infected with E. histolytica. Only 40% of the children infected with E. histolytica were diagnosed microscopically, and the rest were positive by other sensitive methods, such as antigen detection and isoenzyme analysis of cell culture. The microscopic technique could not diagnose many cases of E. histolytica infection, which were positive by antigen detection and isoenzyme analysis of cell culture (36). Several studies show that the detection of E. histolytica antigen in stool by ELISA technique is a more reliable, sensitive, and specific method, faster and easier to perform in many medical laboratories (34, 37-39). The sensitivity and specificity of coproantigen test are evaluated by many studies. el-Hamshary et al. (40), reported that coproantigen ELISA in 93 patients susceptible to amoebiasis was more sensitive and specific than microscopic methods to differentiate between pathogen and nonpathogen Entamoeba species. Baumann et al. (41), showed that 14 out of 15 suspected patients were diagnosed with amoebiasis (93% sensitivity) using coproantigen ELISA kits. Singh et al. (42), reported that the diagnosis of amoebiasis using coproantigen ELISA kits had an 89% sensitivity and 100% specificity. Urdaneta et al. (43), showed that coproantigen ELISA is a more reliable method than the microscopic examination with 98.3% sensitivity and 97.6% specificity. The positive and negative predictive values were 96.2% and 97.6%, respectively, to detect E. histolytica in stool samples.

There was a 100% correlation between the antigen detection kit and the conventional nested PCR results for E. histolytica diagnosis in suspected patients (22, 23, 35). Gharibi et al. (44), presented that among 200 patients with dysenteric diarrhea, 17, 30, and 23 were positive for E. histolytica/E. dispar using microscopic, coproantigen, and PCR techniques, respectively. They reported that the more positive samples were detected by coproantigen ELISA.

5.1. Conclusions

According to the current study findings, there are many E. histolytica misdiagnoses in medical diagnostic laboratories in the studied region, and the rate of E. histolytica infection is very low among children with dysenteric diarrhea. Therefore, useful training of laboratory personnel to promote experience and use of a combination of culture and coproantigen detection techniques are strongly recommended. The small sample size was one of the limitations of the current study; therefore, larger sample sizes should be considered in further studies.

References

  • 1.

    Haque R, Huston CD, Hughes M, Houpt E, Petri WA. Current concepts: amebiasis. N Engl J Med. 2003;(348):1565-73.

  • 2.

    Ximenez C, Moran P, Rojas L, Valadez A, Gomez A. Reassessment of the epidemiology of amebiasis: state of the art. Infect Genet Evol. 2009;9(6):1023-32. [PubMed ID: 19540361]. https://doi.org/10.1016/j.meegid.2009.06.008.

  • 3.

    Forsell J, Koskiniemi S, Hedberg I, Edebro H, Evengard B, Granlund M. Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples. J Med Microbiol. 2015;64(9):1053-62. [PubMed ID: 26296348]. https://doi.org/10.1099/jmm.0.000129.

  • 4.

    Korpe PS, Stott BR, Nazib F, Kabir M, Haque R, Herbein JF, et al. Evaluation of a rapid point-of-care fecal antigen detection test for Entamoeba histolytica. Am J Trop Med Hyg. 2012;86(6):980-1. [PubMed ID: 22665604]. [PubMed Central ID: PMC3366543]. https://doi.org/10.4269/ajtmh.2012.11-0661.

  • 5.

    Nazarian I. Intestinal parasitic infestation in Fars Province, Iran. Z Tropenmed Parasitol. 1973;24(1):45-50.

  • 6.

    Hooshyar H, Rezaian M, Kazemi B, Jeddi-Tehrani M, Solaymani-Mohammadi S. The distribution of Entamoeba histolytica and Entamoeba dispar in northern, central, and southern Iran. Parasitol Res. 2004;94(2):96-100. [PubMed ID: 15293045]. https://doi.org/10.1007/s00436-004-1175-8.

  • 7.

    Kebede A, Verweij JJ, Petros B, Polderman AM. Short communication: Misleading microscopy in amoebiasis. Trop Med Int Health. 2004;9(5):651-2. [PubMed ID: 15117312]. https://doi.org/10.1111/j.1365-3156.2004.01236.x.

  • 8.

    Pillai DR, Keystone JS, Sheppard DC, MacLean JD, MacPherson DW, Kain KC. Entamoeba histolytica and Entamoeba dispar: epidemiology and comparison of diagnostic methods in a setting of nonendemicity. Clin Infect Dis. 1999;29(5):1315-8. [PubMed ID: 10524983]. https://doi.org/10.1086/313433.

  • 9.

    Zebardast N, Gharavi MJ, Abadi A, Seyyed Tabaei SJ, Yegrameh F, Khazan H, et al. Frequency of Intestinal Parasites in Patients With Gastrointestinal Disorders, in Different Parts of Iran During 2012-2013. Int J Enteric Pathog. 2015;3(1):22682.

  • 10.

    Abd-Alla MD, Jackson TF, Gathiram V, el-Hawey AM, Ravdin JI. Differentiation of pathogenic Entamoeba histolytica infections from nonpathogenic infections by detection of galactose-inhibitable adherence protein antigen in sera and feces. J Clin Microbiol. 1993;31(11):2845-50. [PubMed ID: 8263165]. [PubMed Central ID: PMC266142].

  • 11.

    Caballero-Salcedo A, Viveros-Rogel M, Salvatierra B, Tapia-Conyer R, Sepulveda-Amor J, Gutierrez G, et al. Seroepidemiology of amebiasis in Mexico. Am J Trop Med Hyg. 1994;50(4):412-9. [PubMed ID: 8166347].

  • 12.

    Roy S, Kabir M, Mondal D, Ali IK, Petri WJ, Haque R. Real-time-PCR assay for diagnosis of Entamoeba histolytica infection. J Clin Microbiol. 2005;43(5):2168-72. [PubMed ID: 15872237]. [PubMed Central ID: PMCPmc1153781]. https://doi.org/10.1128/jcm.43.5.2168-2172.2005.

  • 13.

    Haque R, Mollah NU, Ali IK, Alam K, Eubanks A, Lyerly D, et al. Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests. J Clin Microbiol. 2000;38(9):3235-9. [PubMed ID: 10970364]. [PubMed Central ID: PMCPmc87365].

  • 14.

    Uslu H, Aktas O, Uyanik MH. Comparison of Various Methods in the Diagnosis of Entamoeba histolytica in Stool and Serum Specimens. Eurasian J Med. 2016;48(2):124-9. [PubMed ID: 27551176]. [PubMed Central ID: PMC4970550]. https://doi.org/10.5152/eurasianjmed.2015.0074.

  • 15.

    Strachan WD, Chiodini PL, Spice WM, Moody AH, Ackers JP. Immunological differentiation of pathogenic and non-pathogenic isolates of Entamoeba histolytica. Lancet. 1988;1(8585):561-3. [PubMed ID: 2894495].

  • 16.

    Safi M, Tavalla M, Mardani M, Afrisham R. Prevalence of intestinal parasitic infections among applicants for health cards attending Ahvaz East Health Center during 2012–2013. Asian Pacific Journal of Tropical Disease. 2016;6(2):151-4. https://doi.org/10.1016/S2222-1808(15)61002-7.

  • 17.

    Yosefi F, Rahdar M, Alavi SM, Amin Samany S. A study on Prevalence of Gastrointestinal Parasitic Infections in HIV (+) Patients Referred to Ahvaz Razi Hospital in 2008-2009. Jundishapur J Microbiol. 2012;5(2):424-426.

  • 18.

    Rafiei A, Rahdar M, Valipour-Nourozi R. Isolation and Identification of Parasitic Protozoa in Sampled Water From the Southwest of Iran. Jundishapur J Health Sci. 2014;6(4).

  • 19.

    Ghasemi E, Rahdar M, Rostami M. Prevalence of Entamoeba histolytica/dispar in drinking water in the city of Shush, Khuzestan Province in 2011. Int.J.Curr.Microbiol.App.Sci. 2015;4(2):582-8.

  • 20.

    Feiz Haddad MH, Maraghi S, Ali SA, Feiz Haddad R, Nasser Zadeh R. Intestinal parasitic infections frequency in referred patients to a large teaching hospital, Khuzestan, Southwest, Iran, 2017. Tropical Biomedicine. 2018;35(4):915-25.

  • 21.

    Haghighi A, Rasti S, Nazemalhosseini Mojarad E, Kazemi B, Bandehpour M, Nochi Z, et al. Entamoeba dispar: genetic diversity of Iranian isolates based on serine-rich Entamoeba dispar protein gene. Pak J Biol Sci. 2008;11(23):2613-8. [PubMed ID: 19630212].

  • 22.

    Solaymani-Mohammadi S, Rezaian M, Babaei Z, Rajabpour A, Meamar AR, Pourbabai AA, et al. Comparison of a stool antigen detection kit and PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar infections in asymptomatic cyst passers in Iran. J Clin Microbiol. 2006;44(6):2258-61. [PubMed ID: 16757634]. [PubMed Central ID: PMC1489450]. https://doi.org/10.1128/JCM.00530-06.

  • 23.

    Haghighi A, Rezaeian M. Detection of serum antibody to Entameba histolytica in various population samples of amebic infection using an enzyme-linked immunosorbent assay. Parasitol Res. 2005;97(3):209-12. [PubMed ID: 15997409]. https://doi.org/10.1007/s00436-005-1418-3.

  • 24.

    Savadkoohi R, Rostaminejad B, Noori NA, Hajian K. Prevalence Of Entamoeba Histolytica In Children With Dysentery (Amirkola Children Hospital, Babol, Iran; 2005-2006). Journal of Babol University of Medical Sciences. 2008;10(5):68-73.

  • 25.

    Sayyari AA, Imanzadeh F, Bagheri Yazdi SA, Karami H, Yaghoobi M. Prevalence of intestinal parasitic infections in the Islamic Republic of Iran. East Mediterr Health J. 2005;11(3):377-83. [PubMed ID: 16602457].

  • 26.

    Karambaigi F, Abdi J, Kourosh Sayehmiri K. Prevalence of Entamoeba histolytica in Iran during 1988 to 2009: Systematic review and meta-analyses. African Journal of Microbiology Research. 2012;6(17):3944-7.

  • 27.

    Battikhi MN. Bloody diarrhoea cases caused by Shigella and amoeba in Jordan. New Microbiol. 2004;27(1):37-47. [PubMed ID: 14964404].

  • 28.

    Kaur R, Rawat D, Kakkar M, Uppal B, Sharma VK. Intestinal parasites in children with diarrhea in Delhi, India. Southeast Asian J Trop Med Public Health. 2002;33(4):725-9. [PubMed ID: 12757217].

  • 29.

    Peruzzi S, Gorrini C, Piccolo G, Calderaro A, Dettori G, Chezzi C. Prevalence of intestinal parasites in the area of Parma during the year 2005. Acta Biomed. 2006;77(3):147-51. [PubMed ID: 17312984].

  • 30.

    Omar MS, Mahfouz AA, Abdel Moneim M. The relationship of water sources and other determinants to prevalence of intestinal protozoal infections in a rural community of Saudi Arabia. J Community Health. 1995;20(5):433-40. [PubMed ID: 8550870].

  • 31.

    Tinuade O, John O, Saheed O, Oyeku O, Fidelis N, Olabisi D. Parasitic etiology of childhood diarrhea. Indian J Pediatr. 2006;73(12):1081-4. [PubMed ID: 17202634].

  • 32.

    Sharma BK, Rai SK, Rai DR, Choudhury DR. Prevalence of intestinal parasitic infestation in schoolchildren in the northeastern part of Kathmandu Valley, Nepal. Southeast Asian J Trop Med Public Health. 2004;35(3):501-5. [PubMed ID: 15689056].

  • 33.

    Dogan N, Oz Y, Kocman NU, Nursal AF. [Comparison of individual differences in the direct microscopic examination in the diagnosis of intestinal parasites]. Turkiye Parazitol Derg. 2012;36(4):211-4. Turkish. [PubMed ID: 23339941]. https://doi.org/10.5152/tpd.2012.51.

  • 34.

    Özer TT, Yula E, Deveci Ö, Tekin A, Durmaz S, Yanık K. Investigation of Entamoeba histolytica in stool specimens by direct microscopic examination and ELISA in a hospital. Dicle Medical Journal / Dicle Tip Dergisi. 2011;38(3):294-7.

  • 35.

    Haque R, Ali IK, Akther S, Petri WJ. Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection. J Clin Microbiol. 1998;36(2):449-52. [PubMed ID: 9466756]. [PubMed Central ID: PMC104557].

  • 36.

    Haque R, Faruque AS, Petri WJ. Entamoeba histolytica and Entamoeba dispar infection in children in Bangladesh. Arch Med Res. 1997;28 Spec No:317-8. [PubMed ID: 9033116].

  • 37.

    Tuncay S, Inceboz T, Over L, Yalcin G, Usluca S, Sahin S, et al. [The evaluation of the techniques used for diagnosis of Entamoeba histolytica in stool specimens]. Turkiye Parazitol Derg. 2007;31(3):188-93. Trukish. [PubMed ID: 17918056].

  • 38.

    Gonzalez-Ruiz A, Haque R, Rehman T, Aguirre A, Hall A, Guhl F, et al. Diagnosis of amebic dysentery by detection of Entamoeba histolytica fecal antigen by an invasive strain-specific, monoclonal antibody-based enzyme-linked immunosorbent assay. J Clin Microbiol. 1994;32(4):964-70. [PubMed ID: 8027351]. [PubMed Central ID: PMCPmc267163].

  • 39.

    Singh A, Houpt E, Petri WA. Rapid Diagnosis of Intestinal Parasitic Protozoa, with a Focus on Entamoeba histolytica. Interdiscip Perspect Infect Dis. 2009;2009:547090. [PubMed ID: 19584941]. [PubMed Central ID: PMC2703877]. https://doi.org/10.1155/2009/547090.

  • 40.

    el-Hamshary EM, el-Shewy KA, Hegazy MM, Zakaria H. Diagnostic potentials of copro-antigen detection based ELISA, compared to microscopy in intestinal amoebiasis. J Egypt Soc Parasitol. 2004;34(2):601-10. [PubMed ID: 15287182].

  • 41.

    Baumann D, Gottstein B. A double-antibody sandwich ELISA for the detection of Entamoeba histolytica antigen in stool samples of humans. Trop Med Parasitol. 1987;38(2):81-5. [PubMed ID: 2888183].

  • 42.

    Singh K, Vinayak VK, Bhasin DK, Ganguly NK. A monoclonal antibody-based test system for detection of Entamoeba histolytica-specific coproantigen. Indian J Gastroenterol. 1999;18(3):104-8. [PubMed ID: 10407562].

  • 43.

    Urdaneta H, Rangel A, Martins MS, Munoz JF, Hernandez M. Entamoeba histolytica: fecal antigen capture immunoassay for the diagnosis of enteric amebiasis by a monoclonal antibody. Rev Inst Med Trop Sao Paulo. 1996;38(1):39-44. [PubMed ID: 8762638]. https://doi.org/10.1590/s0036-46651996000100008.

  • 44.

    Gharibi Z, Kazemi F, Tavalla M. Entamoeba spp. diagnosis in patients with inflammatory diarrhea by staining, copro-antigen ELISA and multiplex PCR methods. Asian Pac J Tropic Dis. 2017;7(10):601-3. https://doi.org/10.12980/apjtd.7.2017D7-137.